Post on 24-Apr-2023
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Enlargeosome, an exocytic vesicle resistant to non-ionic detergents, undergoes endocytosis via
a non-acidic route.
Emanuele Cocucci1,2,3, Gabriella Racchetti1, Paola Podini1, Marjan Rupnik2,
and Jacopo Meldolesi1
1 Vita-Salute University, DIBIT, and San Raffaele Scientific Institute, via Olgettina 58, 20132
Milano, Italy. 2 European Neuroscience Institute Goettingen, Waldweg 33, 37073 Goettingen, Germany. 3 Department of Pharmacology, University of Milan, via Vanvitelli 32, 20133 Milan, Italy.
running title: Enlargeosome membranes and exo/endocytosis.
key words: [Ca2+]i; membrane capacitance; immunofluorescence; non-acidic endosomes; membrane
rafts; cholesterol depletion.
text characters (with spaces): 46.252
Correspondence and Proofs to
Jacopo Meldolesi
DIBIT, San Raffaele Institute
Via Olgettina, 58
20132 Milan, Italy
telephone: 39.02.26432770
email: meldolesi.jacopo@hsr.it
http://www.molbiolcell.org/content/suppl/2004/10/05/E04-07-0577.DC1.htmlSupplemental Material can be found at:
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ABSTRACT
Enlargeosomes, a new type of widely expressed cytoplasmic vesicles, undergo tetanus toxin-
insensitive exocytosis in response to [Ca2+]i rises. Cell biology of enlargeosomes is still largely
unknown. By combining immunocytochemistry (marker: desmoyokin-Ahnak, d/A) to capacitance
electrophysiology in the enlargeosome-rich, neurosecretion-defective clone PC12-27, we show that
1. the two responses, cell surface enlargement and d/A surface appearance, occur with similar
kinetics and in the same low µM [Ca2+]i range, no matter whether induced by photolysis of the
caged Ca2+ compound, NP-EGTA, or by the Ca2+ ionophore, ionomycin. Thus, enlargeosomes
appear to account, at least in large part, for the exocytic processes triggered by the two stimulations;
2. the enlargeosome membranes are resistant to non-ionic detergents but distinct from other
resistant membranes, rich in caveolin, Thy1 and/or flotillin1; 3. cell cholesterol depletion, which
affects many membrane fusions, neither disrupts enlargeosomes nor affects their regulated
exocytosis; 4. the post-exocytic cell surface decline is [Ca2+]i-dependent; 5. exocytized d/A-rich
membranes are endocytized and trafficked along an intracellular pathway by non-acidic organelles,
distinct from classical endosomes and lysosomes. Our data define specific aspects of enlargeosomes
and suggest their participation, in addition to cell differentiation and repair, for which evidence
already exists, to other physiological and pathological processes.
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INTRODUCTION
Regulated exocytosis is commonly envisaged as the final step of regulated secretion, i.e. the
stimulation-induced fusion with the plasmalemma of the membrane of secretory granules and
vesicles, followed by the release of their segregated content (see Gerber and Sudhof, 2002;
Burgoyne and Morgan, 2003 for reviews). Regulated exocytoses, however, are not always
secretory. The ultimate task of many of them is in fact the transfer of membrane patches, or of their
components, from a cytoplasmic compartment to the cell surface. Processes of this type are
widespread among cells.
Many exocytic, non-secretory membrane transfers were first revealed by observations
documenting the stimulation-induced appearance at the cell surface of specific integral membrane
proteins (see for example Sheng and Lee, 2001; Brown, 2003; Bryant et al., 2002). In contrast, in
two fibroblast lines (CHO and 3T3: Coorssen et al., 1996; Ninomiya et al., 1996) and in PC12-27
(Kasai et al., 1999), a neurosecretory clone defective of regulated secretion (Malosio et al., 1999;
Grundschober et al., 2002), the first observations, made by electrophysiological capacitance/patch
clamp assays, consisted in considerable (15-30%) expansions of the plasma membrane in response
to large increases of the cytosolic Ca2+ concentration ([Ca2+]i), induced by photolysis of caged Ca2+
compounds. In PC12-27 these responses start a few hundreds of msec after stimulation and develop
rapidly (t1/2 <1sec), sustained by the tetanus toxin (TeTx)-insensitive exocytosis of small vesicles
(diameter <0.1 µm: Kasai et al., 1999). The latter results, which exclude the involvement of the
TeTx target protein, the vSNARE VAMP2 (Schiavo et al., 1992), differentiate the process from the
regulated exocytoses of neurosecretory cells, such as wild-type (wt) PC12 and chromaffin cells,
which are TeTx-sensitive (Xu et al., 1998; Kasai et al., 1999).
Knowledge about non-secretory exocytosis is not limited to electrophysiology. Seminal cell
biological studies have been carried out by the use of a monoclonal antibody (indicated as Ab)
specific for an exocytic vesicle marker (Borgonovo et al., 2002). The latter, identified as a form of
the high mol wt protein desmoyokin/Ahnak (d/A) (Shtivelman and Bishop, 1993; Hashimoto et al.,
1995) was shown: i. to be expressed by many, but not all types of cells, of both animal tissues and
cultured lines, including those where non-secretory capacitance responses had been reported: CHO,
3T3 and PC12-27; ii. to be localized at rest within vesicles distinct from other cytoplasmic
organelles; and iii. to undergo rapid, TeTx-insensitive transfer to the surface of the cell upon
stimulation with the Ca2+ ionophore, ionomycin. Evidence suggested the d/A-positive vesicles to
have a role in the surface enlargements occurring in important processes, such as cell differentiation
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and plasma membrane repair. Based on these considerations the exocytic vesicles marked by d/A
were given the name of enlargeosomes (Borgonovo et al., 2002).
In our previous study (Borgonovo et al., 2002), the electrophysiological and cell biological
results were assumed to be both due to enlargeosome exocytosis. However, this identification
remained open to question. In fact, the capacitance increases induced by caged Ca2+ photolysis
appeared to require considerable [Ca2+]i rises (Kasai et al., 1999), much larger than those induced
by the ionophore employed in the cell biological studies. Moreover, no comparative results existed
about many aspects of the exocytic responses induced in the same cells by the two types of
stimulation. Therefore, the possibility that the responses were due to two parallel processes sharing
some properties, such as TeTx-insensitivity, could not be excluded, also because knowledge about
the properties of enlargeosomes was still limited.
Here we report about an integrated electrophysiological/cell biological investigation of
enlargeosome exocytosis and the ensuing endocytic process, carried out in the defective PC12-27
clone. Our results, on the one hand, document that the Ca2+-dependence of the responses induced by
photolysis and ionomycin is not as different as previously reported, but rather falls in the same
range; on the other hand, reveal new and unexpected properties of the enlargeosome and of its
exocytic process and demonstrate for the first time the post-exocytic endocytosis of d/A-positive
membranes.
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MATERIALS AND METHODS
Cloned PC12-27, wt PC12, HeLa cells and monoclonal antibodies, IgG2a anti-d/A Ab and
IgG2a anti-chromograninB CIRO, were as in Borgonovo et al. (2002). TeTx and the IgG1 anti-
Lamp1 monoclonal were the gift of C. Montecucco and I. Mellman, respectively. The IgG1 anti-
TGN 38 monoclonal and the anti-EEA1 polyclonal were from Affinity Bio-Reagents, Golden, CO;
the IgG1 anti-transferrin receptor monoclonal, from Zymed, San Francisco CA; the IgG1 anti-G58K
monoclonal, from Abcam, Cambridge U.K.; the anti-caveolin1 polyclonal, and the IgG1 anti-
flotillin1 monoclonal, from BD Biosciences Pharmingen, Heidelberg, Germany; the IgG1 anti-Thy1
monoclonal, from Serotec, Oxford UK; cholera toxin-Alexafluor488, NP-EGTA and Fura-6F, from
Molecular Probes, Eugene, OR; fluorescein isothiocyanate (FITC)-conjugated and
tetramethylrhodamine isothyocyanate (TRITC)-conjugated goat anti-mouse, goat anti-rabbit and
goat anti-mouse IgG subclasses, from Southern Biotechnology Associated, Birmingham, AL.; the
E2-Link Activated Peroxydase kit and the Immunopure Fab kit, from Pierce, Rockford IL;
CypHer5, from Amersham Biosci., Little Chalfont, UK; ionomycin from Calbiochem, Schwalbach,
Germany; other chemicals from Sigma, St. Louis, MO.
Cell cultures
Media were supplemented with 2 mM L-glutamine, 100 U ml-1 penicillin, and streptomycin
(Biowhittaker, Verviers, Belgium). PC12 wt and PC12-27 were grown in Dulbecco’s modified
Eagle’s medium with 10% horse serum (Euroclone, Wetherby, UK) and 5% fetal clone III serum
(Hyclone, Logan, UT); HeLa cells in the same medium without horse serum and with 10% fetal
clone III serum; hybridoma cells in Iscove’s MDM medium with 10% fetal cloneI serum, 5%
macrophage conditioned medium and 50 µM β-mercaptoethanol.
Patch clamp experiments
Patch pipettes were pulled from borosilicate glass capillaries (GC150F-15; WPI, Sarasota,
FA) with a horizontal puller (P-97; Sutter, Novato, CA) to a resistance of 1-4 MΩ in a solution
containing (in mM): 100 KCl, 10 TEACl, 40 KOH/HEPES, 2 Na2ATP, 2 MgCl2, 3.6 CaCl2, 4 NP-
EGTA and 0.5 Fura-6F, pH 7.3. In ionomycin experiments CaCl2 and NP-EGTA were omitted. The
bath contained (in mM): 130 NaCl, 5 KCl, 1 MgCl2, 30 NaOH/HEPES, 4 CaCl2, 6 glucose, pH 7.3.
All recordings were made at room temperature.
Membrane capacitance (Cm) and access conductance (Ga), compensated by Cm and Ga
control, were measured by using a SWAM II C (Celica, Ljubljana, Slovenia), operating at 800 Hz
lock-in frequency, and by applying a sine voltage of 11 mV rms. The phase angle setting was
determined by applying 1-pF pulses and monitoring their projection from the C (signal proportional
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to Cm) to G outputs of the lock-in amplifier. Cm, Ga, membrane current and membrane potential
were recorded unfiltered into a PC via an A/D converter (PCI-6035E, National Instruments, Austin,
TX). WinWCP software (John Dempster, Strathclyde University, UK) was used to acquire and
analyze data. Graphs were drawn using the Sigma Plot (SPSS, Chicago, IL).
[Ca2+]i measurements in flash photolyzed and ionomycin-stimulated cells.
A UV flash from a Xe arc flash lamp (JML-C2, Rapp Opto-Electronic, Hamburg, Germany)
was delivered, through an optical fiber to a 40X fluor oil immersion objective of an Axiovert 200
Zeiss microscope, to whole-cell patch clamped PC12 or PC12-27 loaded with both NP-EGTA and
Fura-6F. The optical pathway included a combination of two mirrors, the first to merge 85% of
flash light and 15% of fluorescence excitation light (M70-85/15 Rapp. Opto-Electronic, Hamburg,
Germany); the second, a dichroic mirror (395 nm), to reflect both lights through the objective to the
cell, with the emitted Fura-6F fluorescence passing back to the photomultiplayer through a 420 nm
filter. The Ca2+ sensor was excited by a monocromator at 380 nm, and the emitted light was
acquired by a Hamamatsu photomultiplier (Till Photometry System, Gräfelfing, Germany); the
signal was recorded after filtering (300Hz, 4-pole Bessel). Ionomycin was applied directly to the
extracellular solution. This implied a delay of the effects of approximately 30 sec that was corrected
in the traces. In each cell [Ca2+]i was calibrated by measuring autofluorescence (in the cell-attached
configuration) and fluorescence (in a resting whole-cell recording) (Kreft et al., 1999).
Differential centrifugation
Suspensions of PC12-27 in 0.32 M sucrose, 5 mM HEPES pH 7.4 and protease inhibitors
were gently homogenized in a cell cracker (Borgonovo et al., 2002), and the homogenates were
centrifuged at 500 rpm for 5 min. The post-nuclear pellet and the final supernatant were obtained by
centrifuging the first supernatant at 40,000 rpm for 60 min.
Resistance to non-ionic detergents
The post-nuclear pellet, carefully resuspended in 0.32 M sucrose, 5 mM HEPES pH 7.4 and
protease inhibitors, was supplemented with Triton X-100 (TX-100), 0.5% final concentration. After
30 min in ice, the preparation was applied to either floatation (Schuck et al., 2003) or top-down
(Gagnoux-Palacios et al., 2003) gradients. In the first, loading was in a 1.23 M sucrose cushion (2
ml) at the bottom of a SW 50.1 tube, covered with 2 ml of 1.10 M and then with 0.15 M sucrose to
volume. In the second, the preparation in 0.32 M sucrose was applied in the same type of tube over
two cushions of 1 and 1.3 M sucrose. After 18 hr at 46,000 rpm, 5 fractions and the pellet were
collected from either gradient and processed for western blotting.
Monolayers of PC12-27 cells in PBS, exposed in the cold to 1% TX-100 as above, were
fixed and processed for immunolabeling.
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Cholesterol depletion
Monolayers in the serum-containing medium were treated with methyl-ß-cyclodextrin (cdx),
3.8 mM, for 10-50 min, then cholera toxin-Alexafluor488 (0.5 g/ml) was added (to monitor its
endocytosis; Wolf et al., 2002) and the incubation was pursued for 10 min. After 5 min treatment
with ionomycin (3 M) or its solvent, cells were fixed and immunolabeled. For SDS-PAGE, cell
processing was similar, but cholera toxin and ionomycin were not added.
SDS-PAGE and Western blotting
Resuspended monolayers were solubilized in ice-cold medium containing (in mM) 50
HEPES, pH 7.5, 150 NaCl, 15 MgCl2, 1 EGTA, 10% glycerol and 1% TX-100, then quickly
centrifuged at 20,000g for 5 min to eliminate nuclei. Protein was assayed with bicinchoninic acid.
For detergent resistance experiments, fixed volumes of the gradient fractions were loaded for SDS-
PAGE; otherwise fixed amounts of protein were loaded. For western blotting, gels transferred to
nitrocellulose filters were first blocked for 1 h with 5% non-fat dry milk in TBS, then incubated for
3 h with the primary antibody diluted in PBS with 3% BSA, washed in TBS (5 fold for 10 min),
incubated for 1 h with the peroxidase-conjugated secondary antibody (1 µg ml-1), washed again in
TBS as above and once in PBS, and developed by chemiluminescence (ECL Western Blotting
Detection, Amersham Biosci, Little Chalfont, U.K.). Signals were acquired by Personal
Densitometer SI and Image Quant (Amersham Biosci, Little Chalfont, U.K.).
Immunofluorescence and Imunoelectron microscopy
For cell surface immunofluorescence, monolayers plated on poly-(L-lysine)-coated
coverslips, at rest or after ionomycin treatment (0.1-5 µM, 0.5-5 min), treated with or without drugs
and/or cholera toxin as indicated in Figure legends, were fixed on ice for 10 min with 4%
paraformaldehyde in PBS, pH 7.4, quenched with 0.1 M glycine and then washed in PBS
containing BSA, 0.3%, and goat serum, 20%. The latter solution was used also for further washes
and to dissolve antibodies. Exposure to the primary antibodies was for 2 h at 22°C, then monolayers
were washed extensively, exposed for 2 h to FITC- or TRITC-conjugated secondary antibodies,
washed, mounted and extensively analyzed. For quantitations, monolayers were processed as above
except that nuclei were labeled with DAPI. For establishing the cell percentage that had undergone
exocytosis in 5 min application of various concentrations of ionomycin, d/A positive and negative
cells were counted under blind conditions; for measuring quantitatively the d/A surface labeling at
rest and after stimulation, the intensity of the signal in individual surface-labeled cells was
established making reference to an arbitrary scale of six values, and the average score ± SE of 30-40
cell groups was calculated and expressed as percentage of the top fluorescence value. For the time-
course of the ionomycin-induced exocytic responses, parallel monolayers were fixed by rapid
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addition of paraformaldehyde (4%, final concentration) at different times after application of 3 µM
ionomycin and processed as above. The labeling intensity of cell groups was established as before.
For whole-cell immunofluorescence the monolayers, fixed and quenched as above, were washed
with the PBS-BSA-goat serum solution with 0.3% TX-100. Treatment with antibodies was as for
surface immunolabeling except that the solution and washing PBS always contained 0.3% TX-100.
In some experiments the cells were processed by a combination of surface and whole-cell
immunolabeling, i.e. they were first surface immunolabeled with Ab, then permeabilized and
whole-cell immunolabeled with other primary antibodies.
Immunofluorescence experiments of endocytosis were carried out as for surface
immunolabeling except that 1-5 µM ionomycin was administered at 37°C in the presence of Ab for
5–30 min. Monolayers were then washed, fixed, quenched, permeabilized and labeled by the
secondary antibody. In some surface and endocytosis experiments the cells, after d/A
immunolabeling and fixation, were washed with the PBS-BSA-goat serum supplemented with 0.3%
TX-100 and then dually labeled for another marker following the whole-cell immunofluorescence
protocol. Dual immunolabeled samples were analyzed quantitatively for co-localization of d/A with
specific markers using the ImageJ software to establish the fraction of pixels dually labeled above a
threshold, according to the formula: (dually labeled)*(d/A-labeled)–1*100.
Immunofluorescent cells were studied using BioRad MRC 1024 and Leica SP2 AOBS
confocal microscopes. For image deconvolution aimed at blur removal and tridimentional cell
reconstruction, optical sections, taken every 150 nm with a wide field microscope on the Delta
Vision system, were analyzed with the soft WoRx Deconvolve software (Applied precision,
Washington DC).
Electron microscopy of immunoperoxidase endocytosis was carried out as for
immunofluorescence except that Ab was conjugated to horseradish peroxidase while fixation was
with 2% glutaraldehyde-4% paraformaldehyde. The diaminobenzidine reaction was carried out
according to Ochs and Press (1992) except that H2O2 (0.03%) was included in the mixture and the
reaction was arrested after 20 min by washing with Tris buffer. Reacted monolayers were washed
with cacodylated buffer, postfixed in 1% OsO4 for 10 min, dehydrated, embedded in Epon and
examined in a Hitachi H7000 electron microscope.
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RESULTS
The clone of the rat pheochromocytoma PC12 cell line employed here, PC12-27 is
advantageous for the present studies: it is rich of enlargeosomes (Borgonovo et al., 2002); it
maintains most of the molecular and structural properties typical of wt PC12, a neurosecretory line
devoid of enlargeosomes, which can therefore be used for reference; it is specifically defective of
the neurosecretion program, in particular it fails to express both types of neurosecretory organelles,
synaptic-like microvesicles (SLMV) and dense granules (DG), as well as the plasmalemma and
soluble proteins instrumental to their exocytic discharge (see Malosio et al., 1999; Grundschober et
al., 2002); it is very poor of lysosome exocytosis (Borgonovo et al., 2002). Therefore, the
participation of lysosomes to the cell surface enlargement induced by stimulation is negligible.
Ca2+-dependence and kinetics of the non-secretory exocytosis
Our first aim was to establish whether in PC12-27 the rapid surface increase induced by
photolysis of a caged Ca2+ compound, revealed by patch clamp capacitance assay, occurs only in
response to the high [Ca2+]i rises (50 µM or above) previously employed by Kasai et al. (1999).
These rises are much higher than those (a few µM only) induced by the Ca2+ ionophore, ionomycin,
which nevertheless are sufficient to trigger enlargeosome exocytosis, as revealed by the surface
appearance of d/A, the enlargeosome marker (Borgonovo et al., 2002).
In order to answer this question we replaced the previously employed caged Ca2+ compound,
dimethoxynitrophenamine tetrasodium salt (DM-nitrophen), with another compound of lower Ca2+
affinity, o-nitrophenyl EGTA (NP-EGTA), whose induced [Ca2+]i changes can be kept low, similar
to those attained with ionomycin (Yang et al., 2002). The capacitance responses induced by
photolysis of NP-EGTA (maximal increases: ~8% above the resting surface area, Fig. 1c) were
investigated in both the neurosecretory wt PC12 and in the defective PC12-27. The responses of the
first were biphasic, composed by an initial small peak, possibly due to the exocytosis of SLMV,
followed by the slower, higher and more persistent increase due to DGs (Fig. 1a). Both these
increases were almost completely blocked by the intracellular perfusion of TeTx (Fig. 1c). In
contrast, in the defective PC12-27 the increases of capacitance were rapid, monophasic (Fig. 1b)
and unaffected by TeTx (not shown). PC12-27 cells stimulated by photolysis of NP-EGTA were
also processed for surface immunofluorescence to reveal enlargeosome exocytosis. As can be seen
in Fig. 1d, a cell fixed with paraformaldehyde a few sec after photolysis appeared already surface-
positive for the specific marker, d/A, documenting that enlargeosome exocytosis had quickly taken
place.
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Fig. 2 shows results obtained in PC12-27 cells treated with ionomycin, 3 µM. With this drug
the [Ca2+]i rises were slow and were followed by delayed increases of capacitance, irregular in their
outline (possibly due to the co-existence of some endocytosis), reaching plateaus within 3-4 min
(Fig. 2a; see also Huang and Neher, 1996). These traces were compared to the quantized surface
immunolabeling responses induced by the ionophore, administered for different times (30 sec to 5
min), followed immediately by quick fixation and immunodecoration of the cells. As can be seen in
Fig. 2b, the increase of the surface d/A signal induced by ionomycin followed a kinetics similar to
that of the capacitance responses. As far as the concentration dependence, d/A became appreciable
at the surface of a few PC12-27 cells already at 0.1 µM ionomycin (average [Ca2+]i around 1.0 µM).
At 3 µM and above the majority of the cells appeared positive (Fig. 2c), most often showing strong
immunolabeling signals (Fig. 2d; see also Borgonovo et al., 2002).
The enlargeosome membrane is resistant to non-ionic detergents
So far, little was known about the enlargeosome membranes. In particular, their resistance to
non-ionic detergents, a property dependent on the cholesterol and phospholipid composition, had
not been investigated. A post-nuclear particulate fraction was therefore re-suspended in 0.5% TX-
100 at 4°C and analysed 30 min later by both floatation (Fig. 3) and top-down (not shown) sucrose
gradient centrifugation, with consistent results. As can be seen in Fig. 3c and d, the markers of
organelles known to be mostly solubilized by TX-100, i.e. transferrin receptor (recycling
endosomes, Fig. 3d) and G58K (Golgi cisternae, Fig. 3c), were recovered almost completely in the
dissolved, non-floated fractions 4 and 5, while the ER chaperone proteins, calreticulin and calnexin
(Fig. 3d), and especially the TGN marker TGN38 (Fig. 3c) exhibited a dual distribution, with
similar recovery in the dissolved and in the floated 2 and 3 fractions (shadowed areas in Fig. 3). In
contrast, well known markers of resistant membranes, caveolin1 (van Deurs et al., 2003) and Thy1
(also known as CD90) (Simons and Toomre, 2000), were recovered mostly (>65%) in the floated
fractions (Fig. 3b) together with only 20% of total protein (Fig.3a). In the case of flotillin1, another
recognized resistance marker (Bickel et al., 1997), the recovery in the dissolved 4 and 5 fractions
(∼30%) was higher, and that in the resistant fractions 2 and 3 (<50%) lower than those of caveolin1
and Thy1 (Fig. 3b). The recovery of d/A, on the other hand, was similar to that of the latter two
resistance markers (Fig. 3a). Consistently, the d/A immunofluorescence pattern was apparently
unchanged by the TX-100 treatment applied to PC12-27 cells before fixation (compare in Fig. 3,
panels f to e), whereas in the same cells the transferrin receptor immunofluorescence was
completely removed by the treatment (not shown).
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The detergent resistance of d/A opened the possibility of enlargeosomes to be one of the
known organelles specific for the resistance markers. Previous studies with anti-TGN38 and anti-
ER markers had already excluded the colocalization of Ab with the corresponding antigens
(Borgonovo et al., 2002). As far as the other markers (Fig. 4a-c; quantitative data in Fig. 4d), an
apparent minor d/A co-labeling was observed in PC12-27 with both Thy1 (Fig. 4b) and flotillin1
(Fig. 4c) whereas with caveolin1 (investigated in HeLa cells, which are also rich of enlargeosomes
(Borgonovo et al., 2002), because the available antibody recognized poorly the fixed rat protein of
PC12-27) the immunolabeling pattern appeared almost completely distinct from that of the
enlargeosome marker, which was similar to that of PC12-27 (Fig. 4a).
Immunolabeling of membrane resistant markers was carried out also in ionomycin-
stimulated cells. In order to focus on the surface-exposed d/A and compare its distribution with that
of the other markers, which are not exposed to the extracellular space, we employed the combined
protocol of surface and whole cell immunolabeling, i.e. fixed cells were first exposed to Ab, then
permeabilized and finally exposed to the primary antibody against one of the three investigated
markers. As can be seen in the images of Fig. 4e-g and in the corresponding quantitative data of
Fig. 4h, no coincidence was observed between exocytized d/A and caveolin1 (Fig. 4e, h). In
contrast, some of the stimulation-induced d/A surface labeling appeared to coincide with Thy1 (Fig.
4f, h) and, especially, with flotillin1 (Fig. 4g, h). Part of this coincidence remained evident after
deconvolution of the images (see the Supplemental Material Video I for a tridimensional rendering
of the surface interaction between Thy1 and d/A).
Cholesterol depletion does not inhibit enlargeosome exocytosis
In view of their detergent-resistance, enlargeosome membranes appear to be rich in
cholesterol. We therefore investigated whether, and to what extent, the structure and function of the
vesicle are affected by incubation of PC12-27 cells with the cholesterol extracting agent, cdx (3.8
mM), for up to 60 min. During the last 10 min of the cdx treatment, cholera toxin-Alexafluor488
was added in order to monitor an independent process, toxin endocytosis (Wolf et al., 2002). In
PC12-27 cells the latter process is inhibited almost completely by 1 hr incubation with cdx
(compare Fig. 5a and c; see also the Supplemental Material Fig. 1), in parallel with the drop of
cholesterol revealed by cell labeling with filipin (not shown). In contrast, endocytosis of the
transferrin receptor is unaffected by cdx (not shown).
Cholesterol-depleted PC12-27 cells exhibited some structural alterations, such as flattening
and/or shrinkage of the cell body with redistribution of the enlargeosomes from the sub-plasma
membrane area to the whole cytoplasm, accompanied by their partial aggregation in one or a few
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large clumps (Supplemental Material Fig. 1, compare panels e,f to d; and video II for a
deconvolution analysis). Within living cells, however, most enlargeosomes were not disrupted, as
documented by the good preservation of the d/A band in western blots prepared from total cell
preparations (Supplemental Material Fig. 1g; see Borgonovo et al., 2002). In contrast, in western
blots prepared from gently homogenized, cdx-pretreated cells (but not from control cells) the d/A
band appeared converted into a ladder (Supplemental Material Fig. 1h). This suggests that the
cholesterol-depleted organelles had not resisted the homogenization insult, releasing the marker
which was then cleaved by cytosolic proteases.
Exocytosis of enlargeosomes was not blocked by cholesterol depletion. Compared to control
cells, where cholera toxin endocytosis was considerable (Fig. 5a, b) and only little surface d/A
labeling was visible before stimulation (Fig. 5a and e), the cdx-treated cells showed a progressive
decrease of cholera toxin endocytosis (Fig. 5c, d; Supplemental Material Fig. 1a-c) accompanied by
an increase of the resting d/A surface signal (Fig. 5c, e) The latter, however, was still reinforced by
ionomycin (3-5 µM), applied for 5 min after 60 min of cdx treatment, to an extent similar to that of
cdx-untreated cells (compare Fig. 5b and d; Fig. 5e). We conclude that cholesterol depletion affects
the traffic of enlargeosome membranes at the surface of resting cells without inhibiting significantly
the responses induced by the Ca2+ ionophore.
Post-exocytic endocytosis
In the previous patch clamp studies, the capacitance increases triggered in PC12-27 cells by
photolysis of DM-nitrophen persisted in all cases until the end of the recordings (Kasai et al., 1999).
Whether this was due to a lack of endocytosis or whether it was an artifactual consequence of the
high [Ca2+]i rises induced in the cells to stimulate exocytosis, remained unclear. In order to
investigate this point, a systematic analysis was carried out in PC12-27 cells stimulated at lower
[Ca2+]i by photolysis of NP-EGTA. Fig. 6a illustrates a photolyzed cell ([Ca2+]i around 4 µM),
representative of an approximately 60% subpopulation of analyzed cells, in which the increased
capacitance remained almost stable during the first 30 sec after photolysis and was largely
unaffected by the application of further photolysis flashes inducing additional [Ca2+]i increases. On
the contrary, the cells in Fig. 6b and c, representative of the remaining sub-population, showed
appreciable declines of the NP-EGTA photolysis-induced capacitance increases, which were
accelerated by further photolysis flashes (Fig. 6c). These observations suggested that, at least in the
second subpopulation, post-exocytic endocytosis is regulated by [Ca2+]i in the 3-5 µM range.
Indeed, in the cells where photolysis had induced relatively low [Ca2+]i rises (average 3.76 ± 0.25
µM; n=5) the subsequent capacitance decreases were slow (~ 30 fF/sec) and only partial during the
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first min (Fig. 6d, e), whereas in those with higher [Ca2+]i rises (average of 4.82 ± 0.24 µM; n=5),
the rate of decline was almost 4 fold faster and the values reached at 0.5-1 min after stimulation
were close to resting (Fig. 6b-e).
In the cells stimulated with ionomycin, on the other hand, a large post-stimulatory
capacitance decline was never observed as long as the recording could be pursued (several min).
Rather, the rate was slow and the decline was only partial. Irregularities of the traces, already
mentioned during the initial capacitance rise, persisted and increased in the cells during decline
(Fig. 6f). Together with the data of Fig. 2a, these results may suggest that, in the course of the
ionomycin treatment, exo- and endocytosis take place concomitantly, with predominance of
exocytosis during the first few min and of endocytosis thereafter.
d/A endocytosis by non-acidic endosomes
The capacitance results of Fig. 6 document that, in a significant fraction of stimulated PC12-
27 cells, post-stimulatory endocytosis takes place within the first min. However they provide no
information about the nature and properties of the endocytic vesicles. In order to investigate
whether the membrane exhibiting the enlargeosome marker, d/A, participates in the endocytosis,
living cell monolayers were first exposed to Ab, its monovalent fragment (Fab-Ab) or an unspecific
antibody applied with or without ionomycin (3 µM) for 0.5, 5 and 30 min at 37°C, then cooled at
4°C, washed extensively and finally fixed. The intracellular distribution of the endocitized
antibodies was revealed by staining with the secondary antibody applied after membrane
permeabilization with TX-100 and staining of nuclei with DAPI. No intracellular immunolabeling
was appreciable in non-stimulated cells incubated for up to 30 min with either the Ab or the control,
anti-chromogranin B antibody (not shown). Likewise, no labeling was observed when the latter
antibody was applied as above, however together with ionomycin (Fig. 7d). In contrast, with Ab
and Fab-Ab, similar patterns were observed, composed of puncta, initially small and distributed
close to the surface of a few cells, later more and more numerous, enlarged and much more frequent
in the cell population (Fig. 7b-c and Fig. 8). We conclude that, in PC12-27 cells, the enlargeosome
marker d/A is involved in the post-stimulatory endocytosis.
Differential properties of the d/A-positive endosomes were investigated by dual
immunofluorescence with respect to specific markers: the early endosome antigen 1 (EEA1, Fig.
8b) and the transferrin receptor (Fig. 8c), known to reside in the two classes of the coated vesicle-
derived endosomes, the sorting and the recycling endosomes, respectively (Maxfield and McGraw,
2004); TGN38 (Fig. 8a) and Lamp-1 (Fig. 8d), the markers of TGN and lysosomes, where
endosomes and endosomal proteins are often addressed. In all analyzed cells the d/A-positive
14
puncta (red) were preferentially distributed to the cytoplasmic layers in the proximity of the cell
surface, with no obvious overlapping with the other markers ( green in Fig. 8a-d). The latter, on the
other hand, exhibited their well known distribution: clustering in juxtanuclear areas for TGN38
(Fig. 8a) and the transferring receptor (Fig. 8c); the superficial cytoplasmic layers for EEA1 (Fig.
8b); a wide scattering throughout the cytoplasm for Lamp1 (Fig. 8d). The lack of co-localization of
these markers with d/A puncta was confirmed by a quantitative analysis of groups of 10 cells
selected at random. In these groups, in fact, apparent coincidence did not exceed average values
(<10%) attributable to the low resolution power of confocal microscopy.
Subsequent experiments were carried out to establish whether the lumen of the d/A-positive
endosomes is acidic, as it is the case of classical endosomes and lysosomes, or whether in contrast it
is neutral. To investigate the problem, Ab was coupled to a pH-sensitive dye, CypHer5, which is
unstained in the neutral and turns red in the acidic environment (Adie et al., 2002). Parallel
experiments were carried out for reference with the dye coupled to the antibody against the
lysosomal marker, Lamp1. Fig. 9a shows PC12-27 cells stimulated for 30 min with ionomycin in
the presence of Ab-CypHer5, subsequently fixed, permeabilized and stained with the FITC-
conjugated secondary antibody. As can be seen these cells exhibited numerous puncta which were
all green. The results indicate that, in the endocytic organelles reached by d/A during the 30 min
incubation, CypHer5 remains unstained, i.e. that the organelle lumen is neutral. In contrast, when
the 30 min ionomycin stimulation was carried out in the presence, not of Ab- but of Lamp1-
CypHer5, the color of puncta was yellow (Fig.9b), as expected by the combination of the green of
the FITC-secondary antibody with the red acquired by CypHer5 in the acidic environment of
lysosomes. Similar results: green puncta with Ab-CypHer5 and yellow puncta with anti-Lamp-
CypHer5, were obtained in further, longer incubation experiments carried out with and without
stimulation by ionomycin. The results obtained in unstimulated PC12-27 cells incubated for 24 hr
with either Ab-CypHer5 or anti-Lamp1-CypHer5 are shown in Fig. 9c and d, respectively. The
neutral lumen, therefore, is not a property of the d/A-positive, post-exocytic endosomes only, but
also of the organelles where the enlargeosome marker is recovered long time after endocytosis.
Taken together with the dual labeling data of Fig. 8, these results confirm that the d/A-rich
endocytic/recycling organelles are distinct from classical endosomes/lysosomes.
The investigation of d/A endocytosis was pursued at the ultrastructural level. Previous
attempts of enlargeosome immuno-electron microscopy had been largely unsuccessful because,
after fixation with even low concentrations of glutaraldehyde, the antigen is poorly recognized by
Ab (Borgonovo et al., 2002). In the present study we focused on the distribution and morphology of
the organelles labeled by peroxidase-coupled Ab applied for 5-30 min to living PC12-27 cells
15
during ionomycin stimulation. With the control monoclonal antibody (against chromograninB,
which is not expressed by PC12-27), administered at the same concentration and for the same times
of Ab, no detectable intracellular labeling was observed (not shown). In contrast, with Ab
cytoplasmic organelles were labeled, although to variable extent. In the intensely positive cells,
weakly labeled vesicles were accompanied by more numerous, distinctly larger (diameters between
80 and 150 nm), and strongly labeled profiles, often of irregular shape (Fig. 10a, c), that could be
clustered in groups in the proximity to plasma membrane (Fig. 10b). Deeper in the cytoplasm the
organelle labeling was intense, concentrated primarily into vacuoles (300-600 nm in diameter),
some of which containing discrete vesicles (Fig. 10c-f). These vacuoles often showed small, coated
bulges, looking as budding vesicles (Fig. 10d, e). In the Golgi/TGN area d/A-positive organelles
were absent.
In the labeling conditions employed, the DAB precipitate was always distributed in direct
contact with the inner face of the organelle limiting membrane. Interestingly, the decoration
appeared not random but distributed according to a pattern composed by rows of puncta aligned at
~40 nm, center-to-center distance from each other (Fig. 10a-e). This decoration pattern might be
related to the structure of the d/A protein, composed by two terminal domains connected by over 30
internal repeats (Shtivelman and Bishop, 1992), each including a site for Ab binding (Borgonovo et
al., 2002).
16
DISCUSSION
The occurrence in various cell types of regulated, non-secretory exocytoses, taking place in
response to large [Ca2+]i increases and inducing considerable surface enlargements, had been shown
several years ago by patch clamp capacitance studies (Coorssen et al., 1996; Ninomiya et al., 1996;
Kasai et al., 1999). More recently, the search for the corresponding vesicles lead to the discovery of
enlargeosomes, identified by their lumenal marker, the high mol wt, peripheral membrane protein,
d/A (Shtivelman et al., 1992; Shtivelman and Bishop, 1993; Hashimoto et al., 1995), which upon
exocytosis remains bound to the cell surface (Borgonovo et al., 2002). So far, however, the
exocytoses revealed by capacitance and those revealed by immunocytochemistry were investigated
separately. On the one hand, some of their properties appeared common: both are due to small
vesicles; both are Ca2+-dependent and insensitive to TeTx; on the other hand, one had been
triggered rapidly (t1/2 <1 sec) by high [Ca2+]i jumps (50 µM and above, Kasai et al., 1999), the other
was much slower and occurred already at low µM [Ca2+]i (Borgonovo et al., 2002). Although very
suggestive, therefore, the possibility that they are due to the same process was still open to question.
The results we have obtained by a combined, capacitance/immunocytochemical
investigation strengthen the single process hypothesis, solving in particular the problem of the
different Ca2+ dependence. By replacing the high affinity caged Ca2+ compound, DM-nitrophen,
with the lower affinity NP-EGTA, we showed that [Ca2+]i rises much lower than those previously
reported, are sufficient to trigger the capacitance responses, i.e. they fall in the same range of those
induced by ionomycin, the Ca2+ ionophore mostly used in the immunocytochemical studies.
Moreover, the d/A exocytic responses induced by the ionophore were shown to develop
concomitantly to slow, but considerable capacitance rises, following the [Ca2+]i responses. It should
be mentioned, however, that since the increase of the cell surface area induced by exocytosis of the
d/A-positive vesicles could not be evaluated quantitatively, we cannot exclude that only part of the
capacitance responses are accounted for by enlargeosomes, the rest being due to other, so far
unknown exocytic organelles, exocytized in parallel and with similar properties.
Our results extend significantly our knowledge about enlargeosomes and their cellular role.
The low µM [Ca2+]i dependence of their exocytosis expands the range of physiological and
pathological processes in which these vesicles could be involved. Some of these processes, cell
differentiation and membrane repair, have already been identified (Borgonovo et al., 2002; Cerny et
al., 2004). Moreover, the enlargeosome membranes were found to be resistant to non-ionic
detergents, a property attributed to the existence, in the plane of their membrane, of small
microdomains (<100 nm in diameter), the rafts, rich in cholesterol and sphingolipids (Pralle et al.,
17
2000; Simons and Toomre, 2000; Anderson and Jacobson, 2002; Parton and Hancock, 2004), where
specific proteins accumulate in a dynamic equilibrium with the other membrane domains
(Kenworthy et al., 2004). Extensive evidence in various organelles and processes (especially the
TGN; endocytosis; constitutive exocytosis) has demonstrated the key role of rafts in membrane
dynamics (fusions, fissions, trafficking and interactions: see the reviews of Nichols, 2003; Helms
and Zurzolo, 2004; Mayor and Rao, 2004). The abundance of rafts, as observed in enlargeosomes,
is not common among the organelles competent for regulated exocytosis, and could therefore play
specific roles at various steps of the new vesicle life, including (in addition to exocytosis, which is
discussed below): generation, presumably at specific domain(s) of the TGN (Maxfield, 2002;
Gleeson et al., 2004; Gkantiragas et al., 2001); traffic and possible interactions, homologous and
possibly also heterologous with other membrane systems. The recognition of the extensive
detergent resistance, the first general property of the enlargeosome membrane so far identified,
could serve in the study of these processes, for example by providing criteria for the isolation and
characterization of the vesicles together with important cues and tools for the interpretation of
future results.
As a direct fall-out of membrane resistance results, we exposed the cells to cholesterol
depletion, believed to disassemble the rafts and to reveal therefore their role in specific processes.
Interestingly, this treatment had been reported to inhibit all types of regulated exocytosis
investigated so far (Lang et al., 2001; Ohara-Imaizumi et al., 2004; Salaun et al., 2004). In PC12-27
cells, depletion was amply effective, as revealed by the blockade of the cholera toxin endocytosis.
Also the enlargeosomes were affected, showing increased fragility, redistribution from the
peripheral areas to the whole cytoplasm, formation of clumps and vacuoles, accumulation of d/A at
the surface of resting cells, possibly due to disturbance of the trafficking to and/or from the plasma
membrane. However, the enlargeosome exocytic responses induced by ionomycin were unchanged,
suggesting their possible independence from the existence of rafts. After the insensitivity to tetanus
toxin, this appears another property that distinguishes the regulated exocytosis of enlargeosomes
from those of other exocytic organelles.
The proposal of the enlargeosome as a new type of organelle was initially based on its
distinction from the classical cytoplasmic structures, the ER, Golgi complex, TGN, sorting and
recycling endosomes, lysosomes, glut4-rich vesicles and constitutive secretory vesicles. The
detergent resistance of the enlargeosome membrane opened now the possibility of a link to the other
detergent-resistant organelles, in particular to caveolin-rich vesicles, caveosomes and other known
raft-rich vesicles trafficking between the TGN and the plasma membrane. Of the three raft markers
investigated, however, caveolin1 was found to lack any co-localization with d/A whereas Thy1 and
18
also flotillin1, which is localized not in a single type of membrane but is distributed to various types
(Morrow et al., 2002; Kokubo et al., 2003), exhibited only a low degree of co-localization. These
results exclude the identification of enlargeosomes with organelles specific for the three markers. A
considerable co-localization of d/A with flotillin1 became apparent at the cell surface after
stimulation of exocytosis. Since, however, the resolution of confocal images is low compared to the
size of the raft microdomains, the significance of the observation, in particular whether it was due to
real intermixing of the exocytized d/A-rich membrane domains with those rich in flotillin1, remains
to be established.
A property of the enlargeosome system that so far was completely unknown is endocytosis.
In the previous patch clamp and immunocytochemical studies (Ninomiya et al., 1996; Kasai et al.,
1999; Borgonovo et al., 2002; Cerny et al., 2004), the process had not been specifically
investigated. Now we have found that a low rate endocytosis of d/A-rich patches occurs even in
resting cells, presumably as a consequence of spontaneous enlargeosome exocytic events; and that
intense endocytosis follows the stimulation-induced exocytic responses. Also in the stimulated
cells, however, endocytosis is markedly asynchronous: it is appreciable within one or a few min
from the stimulation only in a fraction of cells, whereas in others it requires longer times in order to
become well appreciable. For obvious technical reasons only the cells of the first group could be
studied by both patch clamping and immunocytochemistry, while with the others only the second
experimental approach could be employed.
Several aspects of the enlargeosome endocytosis appear of interest. Among the PC12-27
cells investigated by patch clamping those that responded to stimulation with [Ca2+]i rises > 4.5 µM
exhibited capacitance decrease rates several fold faster than those that had reached values <4 µM.
The enlargeosome endocytosis appears therefore to be regulated, a property that, in other cells and
secretory systems, has already attracted great interest (Sankaranarayanan and Ryan, 2001; Sorkin
and Von Zastrow, 2002; Maxfield and McGraw, 2004). Moreover, the intracellular vesicles positive
for d/A maintained a neutral pH in their lumen. This result excludes the involvement of the
endosomes generated from coated pits and vesicles, characterized by their classical acidic lumen
(Maxfield and McGraw, 2004), however it appears insufficient for the identification of the
organelles involved. Various types of non-acidic endosomes have in fact been described, trafficking
independently or coordinately with each other and with acidic endosomes along multiple
intracellular pathways (Nichols and Lippincott-Schwartz, 2001; Nabi and Le, 2003; Nichols, 2003;
Parton and Richards, 2003; Massol et al., 2004). Finally, a sequence of the events occurring within
30 min after the generation of the d/A-positive endocytic vesicles, could be deduced from the
ultrastructural study of cells that had internalized Ab in the course of ionomycin stimulation. Early
19
vesicles, relatively small and moderately immunolabeled, developed into more heavily labeled
organelles, i.e. larger vesicles and then vacuoles with a few segregated vesicles in their lumen and
some vesicles budding from their cytosolic surface. Interestingly, the TGN appeared completely
excluded from the d/A-positive endocytic pathway. Further work is needed to investigate the
numerous issues still open in the field, in particular to identify the nature of the d/A-positive
endosomes; to map in more detail their intracellular pathway; and to clarify the possible role of this
recycling system in the regeneration of enlargeosomes competent for regulated exocytosis
In conclusion, the enlargeosomes that emerge from the present study are significantly more
detailed than those known until now (Borgonovo et al., 2002). Working on the PC12-27 cell model
we have shown that this new type of vesicle does indeed participate in the regulated exocytic
responses and does account, at least in large part, for the surface enlargements revealed by patch
clamp capacitance, induced by both photolysis of caged Ca2+ compounds and the Ca2+ ionophore,
ionomycin; that its membrane, resistant to non-ionic detergents, is different from other membranes
sharing this property; that cholesterol extraction does not block its exocytosis; that after exocytosis
its membrane is recycled by a non-acidic form of endocytosis. Enlargeosomes appear therefore to
have a specific profile, compatible with a wide role in physiology and possibly also in pathology. In
addition to their intrinsic interest, these results promise to be instrumental for future studies on
multiple aspects of the enlargeosome cell biology that are still unknown, including the biogenesis
and intracellular transport of the organelles, the molecular mechanisms of their exocytic membrane
fusion, and the nature and pathways of their endocytic system.
20
ACKNOWLEDGEMENTS
This work was supported by grants from the EU (Growbeta n. QLG3-CT2001-02233 and Apopis
LSHM-CT-2003-503330), the Italian MIUR (Cofin 2001 and 2002; FIRB), Telethon and the Italian
Natl. Research Council (CNR: Physiopathology of the Nervous System and Functional Genomics).
During part of the work Emanuele Cocucci was a EU Marie Curie fellow at the European
Neuroscience Institute, ENI, Göttingen Germany. We thank Erwin Neher for support, Evelina
Chieregatti and Michela Matteoli for suggestions and criticisms.
21
ABBREVIATIONS USED IN THIS PAPER:
Ab, mouse monoclonal antibody raised against d/A; [Ca2+]i, cytosolic concentration of free calcium;
cdx, methyl-β-cyclodextrin; d/A, desmoyokin/Ahnak; DG, dense granule; DM-nitrophen,
dimethoxynitrophenamine tetrasodium salt; EEA1, early endosomal antigen 1; fF, femtoFarad, unit
of capacitance; FITC, fluorescein isothiocyanate; TRITC, tetramethylrhodamine isothyocyanate;
Glut4, glucose transporter4; iono, ionomycin; Lamp1, lysosomal membrane glycoprotein1; NP-
EGTA, o-nitrophenyl EGTA; PC12, rat pheochromocytoma cell line; SLMV, synaptic-like
microvesicle; TeTx, tetanus toxin; TGN, transGolgi network; TX-100, Triton X-100; VAMP2,
vesicle associated membrane protein2, otherwise called synaptobrevin; wt, wild-type.
22
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28
LEGENDS FOR FIGURES
Fig. 1. NP-EGTA photolysis-induced exocytosis in wt PC12 and PC12-27 cells.
Panels a and b show the [Ca2+]i increases and the ensuing plasma membrane capacitance responses
induced by photolysis of the caged Ca2+ compound in representative wt PC12 and PC12-27 cells,
respectively. The biphasic capacitance trace of the wt PC12 (panel a) is probably accounted for by
the rapid exocytosis of SLMVs followed by the slow exocytosis of DGs; the rapid response of the
PC12-27 cell (panel b) may be sustained, entirely or in part, by the exocytosis of enlargeosomes. In
agreement with this possibility, PC12-27 cells fixed a few sec after photolysis and then
immunolabeled, show an intense surface positivity for d/A (panel d). Panel c shows that, on the
average, the maximal capacitance increase (expressed as % of the cell surface area at rest) was
approximately the same in wt and defective PC12 cells, and that in wt PC12 this response was
blocked by tetanus toxin (4 µM inside the pipette, Xu et al., 1998). The bars in panel d and in all
immunofluorescence panels of the subsequent Figures correspond to 10 µm.
Fig. 2. Ionomycin-induced exocytosis in PC12-27 cells.
Panel a shows the increase of capacitance that follows the [Ca2+]i rise in a representative cell
exposed to ionomycin (3 µM, arrow). Notice the slow rate of both responses and the irregular
outline of the capacitance trace, which could be due to concomitant membrane endocytosis. The
capacitance bar of 500 fF corresponds to ∼2.5% of the initial cell surface area. Panel b shows the
time-course of the d/A surface appearance, given for each time-point as the average fluorescence
intensity in a population of 30-40 cells exposed to ionomycin for the indicated times, then rapidly
fixed and surface immunolabeled without permeabilization. Notice that the curve of this panel,
expressed in % of the top value of the scale, resembles roughly the capacitance trace of panel a. The
last point of the trace corresponds to 300 sec of stimulation. Panel c shows the concentration-
dependence of the ionomycin-induced d/A surface appearance, expressed as the % of positive cells
in a population of scanned PC12-27. In the positive cells the intensity of the surface fluorescence
signal did not remain the same but increased considerably as a function of the ionomycin
concentration, in parallel to the % cell positivity, reaching high values such as those of panel d,
which shows cells exposed to 3 µM ionomycin for 5 min.
Fig. 3. Effects of TX-100 on the distribution of d/A in a floatation gradient, compared to other
membrane markers, and on the d/A immunolabeling of PC12-27 cells.
29
The fractions of the floatation gradient (panels a-d) were analyzed by western blotting. Of the
fractions, P is the pellet, 5 and 4 include the components solubilized by TX-100, 3 and 2
(shadowed) are the detergent-resistant fractions, and 1 is the top, 0.15 M sucrose layer. The SDS-
PAGE slots of all lanes except 1were loaded with 100 µl samples of the fractions recovered from
the gradient. Values are given as % recovery of the markers in the various fractions. The traces in
panel a refer to d/A and total protein (tp); those in b to caveolin (cav), Thy1 and flotillin1 (flot);
those in c to the Golgi (G58K) and TGN (TGN38) markers; those in d to the transferrin receptor
(TFR), and the endoplasmic reticulum proteins, calnexin (Cnx) and calreticulin (CR). Panels e and f
illustrate the d/A immunolabeling of PC12-27 cells fixed before (panel e) and after (panel f)
treatment with TX-100 (1%, 30 min at 4°C), and then processed for whole-cell
immunofluorescence.
Fig. 4. Dual immunolabeling of HeLa and PC12-27 cells for d/A (red) and raft markers (green).
Panels a-c refer to HeLa (a) and PC12-27 (b, c), investigated by whole-cell immunolabeling while
at rest. d/A puncta, localized primarily in the superficial layers of the cytoplasm, show very little
apparent co-localization with caveolin1 (panel a), localized primarily at the cell surface. With Thy1
(panel b) and, especially, with flotillin1 (panel c), which are distributed to the whole cytoplasm, the
apparent co-localization is still low but more pronounced than with cavolin1. The flotillin1
immunolabeling of the nucleus is an artifact. In panel d, the apparent co-localization of the three
raft markers with d/A in the cytoplasm of groups of 10 resting cells is given in quantitative terms.
The HeLa cells of panel e and the PC12-27 cells of panels f and g were stimulated with ionomycin
(3 µM, 5 min). After fixation they were first surface immunolabeled for d/A, then permeabilized
and whole-cell labeled for the other markers. Under these conditions, therefore, apparent co-
localization can be observed only at the cell surface. Notice that such a co-localization of d/A with
caveolin1 (panel e) is very low, with Thy1 (panel f) is larger but variable, while with flotillin1
(panel g) it is considerable, as confirmed by the quantitative analyses of panel h, which refers only
to the cell surface. For the Thy1-d/A surface co-localization see also the Supplemental Material
Video I, showing a tridimensional rendering.
Fig. 5. Effects of cholesterol depletion on enlargeosome exocytosis.
PC12-27 cells, pretreated (panels c, d) or not (panels a, b) with cdx for 60 min, were all exposed to
cholera toxin-Alexa488 (green) during the last 10 min of the pretreatment, followed (panels b, d) or
not (panels a, c) by ionomycin stimulation (3 µM, 5 min). After fixation the cells were surface
immunolabeled for d/A (red). Notice that in the cells not treated with cdx (panels a, b) the
30
endocytosis of cholera toxin (green) was intense. The surface d/A labeling was inappreciable in the
cells in panel a, as in all resting PC12-27 cells (see Borgonovo et al., 2002), and became clearly
evident after ionomycin (red and yellow labeling of panel b). After cholesterol depletion (panels c,
d), the general PC12-27 phenotype was altered, with flattening and or/shrinkage of the cell body.
Endocytosis of cholera toxin was blocked (lack of green labeling). A clear surface d/A signal (red)
was visible already in the resting cells (panel c), and was increased by ionomycin to levels similar
to those of stimulated controls (compare panel d to b). Panel e summarizes quantitatively (as in Fig.
2b) the surface d/A labeling results obtained in groups of 10 cells from the populations illustrated in
panels a-d.
Fig. 6. Time-course of capacitance after the stimulation-induced rise in PC12-27 cells: Ca2+-
dependence of endocytosis.
The three representative cells in a-c show jumps of [Ca2+]i to about 4 µM induced by NP-EGTA
photolysis causing parallel capacitance rises of ∼ 600-2000 fF. In the cell of panel a the increased
capacitance remains largely stable, insensitive to further photolysis-induced [Ca2+]i spikes. This cell
belongs to the subpopulation in which endocytosis is delayed. In contrast, panels b and c show cells
of the subpopulation in which endocytosis begins during the first min. Notice that the rate of the
process is faster in c, where subsequent photolysis spikes prevent the decrease of the [Ca2+]i. The
data in d and e illustrate the Ca2+-dependence of the endocytic process. Compared to a group of 5
cells reaching maximal [Ca2+]i levels < 4 µM (average 3.76 ± 0.25 µM), 5 cells reaching levels >
4.5 µM (average 4.82 ± 0.24 µM ) exhibit endocytic rates over 4 fold faster (panel d). Therefore, 30
sec after stimulation the average capacitance values of these cells correspond to an 82% decrease
from the exocytic peak (panel e), i.e. they have returned near the resting values, whereas the
average values of the lower [Ca2+]i group have decreased of only 38%. Panel f shows the time-
course of the capacitance in a representative cell stimulated with ionomycin (3 µM) administered at
the arrow. Notice the irregularities of the trace which declines slowly and remains away from the
resting values for the whole recording time (several min).
Fig. 7. d/A endocytosis in ionomycin-stimulated PC12-27 cells.
The panels show the cellular distribution of Ab applied to living cells together with ionomycin for
0.5 (panel a), 5 (panel b) and 30 (panel c) min. Notice that at the early time point only a small
fraction of cells shows intracellular accumulation of d/A-positive puncta. Later on the number of
positive cells increases and the number and intensity of their puncta also increase, reaching
considerable apparent size at 30 min. With a control monoclonal antibody, against a protein not
31
expressed by PC12-27 cells (chromograninB), incubated with ionomycin (panel d) or without (not
shown), no intracellular labeling was observed.
Fig. 8. Endocytic organelles positive for d/A are distinct from the TGN, classical endosomes and
lysosomes.
The panels show groups of PC12-27 cells exposed to ionomycin (3 µM) together with Ab (red) for
30 min, then fixed and whole-cell labeled (green) for the TGN marker TGN38 (panel a); the sorting
endosome marker EEA1 (panel b); the recycling endosome marker transferrin receptor (panel c)
and the lysosome marker Lamp1 (panel d). Merging of the images of the dually labeled cells
reveals only very low levels of apparent coincidence, documenting a lack of co-localization of d/A
with the investigated markers. Notice also that the overall distribution of the d/A-positive
endosomes in the cytoplasm is mostly peripheral, not only with respect to the TGN and recycling
endosomes, but also to sorting endosomes and lysosomes. The green staining of nuclei in panel b is
an artifact of the polyclonal anti-EEA1 antibody used.
Fig. 9. Distribution of either CypHer–Ab or CypHer–anti-Lamp1 applied to PC12-27 cells during
stimulation with ionomycin for 30 min (panels a, b) or while resting for 24hr (panels c, d).
Notice that the organelles where Ab and anti-Lamp1 antibody accumulate exhibit different color
labeling because of their different lumenal pH. The d/A positive puncta (panels a, c) are all green,
due to the Ab immunolabeling only. In these puncta, therefore, CypHer is unstained, i.e. the lumen
of the organelle is neutral. In contrast, the Lamp1-positive organelles, the lysosomes, are yellow
(panels b, d), the color resulting from the merge of the green of the Lamp1 immunolabeling with
the red that CypHer acquires in the acidic environment of the lumen.
Fig. 10. Ultrastructure of d/A-positive endocytic organelles decorated by Ab-peroxidase.
PC12-27 cells were incubated with Ab-peroxidase together with ionomycin as in Fig. 7. Various
forms of the d/A positive organelles are shown in the panels. In a 3 positive organelles, 100-300 nm
in diameter, are distributed individually in the sub-plasmalemma area, at 1-1.5 µm distance from
each other; in b small (80-100 nm), lightly labeled profiles are clustered in the proximity of the
plasma membrane together with larger, more intensely labeled vesicles; panel c shows at the top a
small positive vesicle, with an organelle of 150 nm diameter and a larger irregular vacuole
containing a few segregated vesicles located in the proximity, but not inside the Golgi/TGN area.
Even larger, irregular vacuoles containing vesicles and with coated bulges, likely corresponding to
budding vesicles (asterisks), are shown in d and e. The latter panel includes also another positive
32
organelle and a vesicle. Finally, panel f shows an enlargement of the vacuole of panel e illustrating
the distribution of the DAB precipitate apposed to the lumenal face of the limiting membrane.
Examples of the periodic pattern of peroxidase labeling of the vesicle and vacuole lumenal
membrane surface are visible in all panels. In contrast to the d/A-positive organelles, the coated pits
and vesicles (arrows and arrowheads in panels c and e) are all d/A negative. The bars in the panels
correspond to 0.5 µm, except panels d and f, where they correspond to 0.2 µm. GC=Golgi complex.
Supplemental Material Fig. 1. Effects of cholesterol depletion on the endocytosis of cholera toxin,
and on the distribution of d/A.
Panels a-c and d-f illustrate the same PC12-27 cells labeled by either cholera toxin-Alexafluor488
or whole-cell d/A immunofluorescence, respectively. Endocytosis of cholera toxin was investigated
by a ten min exposure of the cells to the toxin, to monitor the inhibitory effect of cholesterol
depletion. At time 0 of cdx treatment endocytosis was considerable (panel a); after 30 min it was
only moderately decreased (panel b) and after 60 min it was almost completely blocked (panel c).
During the same treatment, the endocytosis of the transferrin receptor was unchanged (not shown),
the d/A immunolabeling redistributed from the external layers (panel d) to the whole cytoplasm
(panels e and f), while the cells became round and flattened (see Supplemental Material Video II for
a tridimensional rendering). Panel g compares the d/A western blots of cells incubated for 60 min
either without (left) or with (right) cdx and then directly processed. Notice that the recovery of the
protein in the typical, high mol wt band was unaffected by cholesterol depletion. Panel h shows d/A
western blots prepared from the same cell populations of panel g, which however before processing
were gently homogenized and centrifuged to isolate the post-nuclear supernatants. Notice the
almost complete preservation of the d/A band in the controls (left) and its conversion into a
proteolytic ladder in the preparation from cdx-treated cells (right), documenting a fragility of
enlargeosomes to the mechanical insults of homogenization/centrifugation, induced by cholesterol
depletion.
Supplemental Material Video I. Tridimensional rendering of the surface co-distribution of d/A
and Thy1.
A PC12-27 cell stimulated with ionomycin (3 µM, 5 min) was fixed, surface immunostained for
d/A (red) and then whole-cell labeled for Thy1 (green). Notice that a fraction of the surface
33
distributed d/A puncta coincide with the Thy1 green puncta (yellow), suggesting a partial
intermixing of the membranes marked by the two antigens after enlargeosome exocytosis.
Supplemental Material Video II. Tridimensional rendering of two PC12-27 exposed to cholera
toxin-Alexa488 and then whole-cell immunolabeled for d/A.
Video a shows a control cell: notice the green puncta due to endocytosis of cholera toxin and the
preferential distribution of d/A puncta (red) in the superficial layers of the cytoplasm.
Video b shows a cell incubated for 60 min with cdx. Notice that green puncta are no longer visible
because the toxin endocytosis is inhibited after cholesterol extraction. Also the d/A distribution is
altered, with apparent formation of clumps below the plasmalemma, probably composed by
aggregates of puncta, which however remain indistinct from each other in the present exposure of
the video.